New enzymatic process for production of modified hop products

ABSTRACT

The present invention relates to a process for producing a beer bittering agent via enzyme catalyzed bioconversion of hop-derived isoalpha acids to dihydro-(rho)-isoalpha acids and to the novel enzyme catalysts which may be employed in such a process.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith under 37 C.F.R. §1.821 in a computer readable form (CRF) via EFS-Web as file nameKALSEC_76_US_Sequence_Listing_26_Sept_2019.txt is herein incorporated byreference. The electronic copy of the Sequence Listing was created on 26Sep. 2019.

FIELD OF THE INVENTION

The present invention relates to a process for producing a beerbittering agent via enzyme catalyzed bioconversion of hop-derivedisoalpha acids to dihydro-(rho)-isoalpha acids and to the novel enzymecatalysts which may be employed in such a process.Dihydro-(rho)-isoalpha acids have superior characteristics which improveutility as a beverage additive. Consumers may preferdihydro-(rho)-isoalpha acids produced via this process, which does notrequire the use of harsh chemical reagents and which utilizes enzymeswhich may be naturally occurring.

BACKGROUND OF THE INVENTION

Traditional methods of bittering beer use whole fresh hops, whole driedhops, or hop pellets added during the kettle boil. Hop extracts made byextracting hops with supercritical carbon dioxide, or isomerized hoppellets, made by heating hops in the presence of a catalyst are morerecent bittering innovations that have also been adopted by brewers. Hoppellets can also be added later in the brewing process and in the caseof dry hopping, hops are added to the finished beer prior to filtration.These methods suffer from a poor utilization of the bittering compoundspresent in the hops, which impacts the cost unfavorably. Beer or othermalt beverages produced in this manner are unstable to light and must bepackaged in dark brown bottles or cans or placed to avoid the lightinduced formation of 3-methyl-2-butene-1-1-thio (3-MBT) which gives apronounced light-struck or skunky aroma. Placing bottles in cardboardboxes or completely wrapping them in light-proof or light-filteringpaper, foil, or plastic coverings is another expensive method ofprotecting these beverages from light-struck flavor and aroma.

Bitterness in traditionally brewed beer is primarily derived fromisoalpha acids. These compounds are formed during the brewing process bythe isomerization of the humulones, which are naturally occurringcompounds in the lupulin glands of the hop plant. A consequence of thisis, given the natural instability of the isoalpha acids towardsphotochemical reactions in beer, a beverage prone to the formation oflight-struck or skunky flavor and aroma.

Fully light stable beers or other malt beverages can be prepared usingso-called advanced or modified hop acids. Beers made using thesebittering agents can be packaged in non-colored flint glass bottleswithout fear of forming skunky aromas. Dihydro-(rho)-isoalpha acids arereduction products of isoalpha acids which are light stable. To date,these compounds have not been found in nature. Traditionally, theportion of the isoalpha acids which is responsible for thephotochemistry has been altered by reduction of a carbonyl group usingsodium borohydride.

Sodium borohydride is an inorganic compound that can be utilized for thereduction of ketones. It is extremely hazardous in case of skin contact,eye contact, inhalation, or ingestion, with an oral LD50 of 160 mg/kg(rat). Sodium borohydride is also flammable, corrosive, and extremelyreactive with oxidizing agents, acids, alkalis, and moisture (SodiumBorohydride; MSDS No. S9125; Sigma-Aldrich Co.: Saint Louis, Mo. Nov. 1,2015.

Consumers are increasingly expressing a preference for natural materialsover synthetic or semi-synthetic ones. Thus, a need exists not only toprovide compositions employing natural materials as bittering agents forbeer and other beverages, but also processes for more natural productionof said materials.

Biocatalytic production is an emerging technology which provides highlyselective, safe, clean, and scalable production of high value compounds.Biocatalytic production relies on naturally occurring enzymes to replacechemical catalysts.

Enzymes are naturally occurring proteins capable of catalyzing specificchemical reactions. Enzymes exist in nature that are currently capableof replacing chemical catalysts in the production of modified hopbittering compounds (Robinson, P. K., Enzymes: principles andbiotechnological applications. Essays Biochem 2015, 59, 1-41.).

Humulone is a natural secondary metabolite that would be exposed tofungi and bacteria cohabitating with the plant, Humulus lupulus. It ispossible that soil- and plant-dwelling fungi and bacteria possessenzymes capable of modifying humulone for detoxification or scavengingpurposes. Additionally, organisms may have evolved enzymes to modifyhumulone-like molecules, but because of promiscuous activity, theseenzymes possess activity against the compounds of interest, isoalphaacids (Hult, K.; Berglund, P., Enzyme promiscuity: mechanism andapplications. Trends Biotechnol. 2007, 25 (5), 231-238; Nobeli, I.;Favia, A. D.; Thornton, J. M., Protein promiscuity and its implicationsfor biotechnology. Nat. Biotechnol. 2009, 27 (2), 157-167.).

Enzymes which catalyze oxidation/reduction reactions, that is transferof hydrogen and oxygen atoms or electrons from one substance to another,are broadly classified as oxidoreductases. More specifically, enzymesthat reduce ketone groups to hydroxyl groups are known as ketoreductasesor carbonyl reductases and depend on the supplementation of an exogenoussource of reducing equivalents (e.g. the cofactors NADH, NADPH).Consistent with the existing naming of the enzymes characterized herein,the enzymes will be referred to as a “ketoreductases”.

The cost of expensive cofactors (NADH, NADPH) can be reduced byincluding additional enzymes and substrates for cofactor recycling, forexample glucose dehydrogenase and glucose, or by utilizing aketoreductase that is also capable of oxidizing a low-cost and naturalfeedstock, such as ethanol.

Abundant precedence exists for the utility of enzymes in brewing andtheir favorable influence on the final character of beer (Pozen, M.,Enzymes in Brewing. Ind. Eng. Chem, 1934, 26 (11), 1127-1133.). Thepresence of yeast enzymes in the natural fermentation of beer is knownto produce compounds that affect the flavor and aroma of the finalbeverage (Praet, T.; Opstaele, F.; Jaskula-Goiris, B.; Aerts, G.; DeCooman, L., Biotransformations of hop-derived aroma compounds bySaccharomyces cerevisiae upon fermentation. Cerevisia, 2012, 36,125-132.). Exogenously added enzymes provide a variety of improvementsto the brewing process, such as reduced viscosity, increased fermentablesugars, chill-proofing and clarification (Wallerstein, L. (1947)Bentonite and Proteolytic Enzyme Treatment of Beer, U.S. Pat. No.2,433,411.; Ghionno, L.; Marconi, O.; Sileoni, V.; De Francesco, G.;Perretti, G., Brewing with prolyl endopeptidase from Aspergillus niger:the impact of enzymatic treatment on gluten levels, quality attributes,and sensory profile. Int. J. Food Sci. Technol, 2017, 52 (6),1367-1374.). Additionally, hop extracts have been specificallypretreated with enzymes for modifying hop-derived aroma compounds (Gros,J.; Tran, T. T. H.; Collin, S., Enzymatic release of odourantpolyfunctional thiols from cysteine conjugates in hop. J. Inst. Brew.2013, 119 (4), 221-227.).

Prior to the present invention, however, enzymes capable of catalyzingthe reduction of isoalpha acids to dihydro-(rho)-isoalpha acids have notbeen observed in nature, and thus have not been described in theliterature. The process disclosed herein represents a novel enzymaticreaction.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a process forenzymatic production of dihydro-(rho)-isoalpha acids, a modified versionof natural bittering agents derived from the hop plant. The presentprocess is designed to replace current production processes whichutilize the chemical reagent, sodium borohydride. It is a further objectof the invention to provide novel enzyme catalysts which may be employedin such a process.

SUMMARY OF THE INVENTION

The present invention relates to a process that can be scaled up toindustrial levels for bioconversion of iso-alpha acids intodihydro-(rho)-isoalpha acids, which can then be used as a naturallyderived and light stable bittering agent in beverages.

One aspect of the present invention is a process for the high-yieldbioconversion of iso-alpha acids into dihydro-(rho)-isoalpha acidsutilizing a ketoreductase enzyme or a microorganism expressing a genethat encodes said ketoreductase.

A further aspect of the invention relates to such a process forproduction of dihydro-(rho)-isoalpha acids, wherein the process iscarried out in an aqueous system with mild temperature and pHconditions, making it an environmentally benign manufacturing process.

In an embodiment of the invention, bioconversion of isoalpha acids todihydro-(rho)-isoalpha acids comprises the addition of purifiedketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acidsfollowed by incubation until the desired yield is obtained.

In another embodiment of the invention, bioconversion of isoalpha acidsto dihydro-(rho)-isoalpha acids comprises the addition of purifiedketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids inthe presence of isopropanol for cofactor recycling, followed byincubation until the desired yield is obtained.

In a further embodiment of the invention, the concentration of isoalphaacids, i.e. the substrate, is maximized to increase the volumetricproductivity of the bioconversion.

In a further embodiment of the invention, the concentration of thecofactor NADPH or NADP in the mixture is minimized to improve theeconomics of the bioconversion.

In an embodiment of the invention, bioconversion of isoalpha acids todihydro-(rho)-isoalpha acids comprises the addition of purifiedketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids inthe presence of another enzyme (such as glucose dehydrogenase) forcofactor recycling, followed by incubation until the desired yield isobtained.

In another embodiment of the invention, bioconversion of isoalpha acidsto dihydro-(rho)-isoalpha acids comprises the addition of a whole cellbiocatalyst to a mixture of isoalpha acids followed by incubation untilthe desired yield is obtained, wherein the whole cell biocatalyst is animmobilized microorganism expressing the gene which encodes aketoreductase.

In another embodiment of the invention, bioconversion of isoalpha acidsto dihydro-(rho)-isoalpha acids comprises the feeding of isoalpha acidsto a growing microorganism expressing the gene which encodes aketoreductase.

In another embodiment of the invention, bioconversion of isoalpha acidsto dihydro-(rho)-isoalpha acids comprises the addition of thermostableketoreductase enzyme to an extract of alpha acids wherein heat isapplied, and the mixture is incubated until the desired yield ofdihydro-(rho)-isoalpha acids is achieved.

The present invention also relates to novel enzyme catalysts which maybe utilized in the process of the invention as defined above.

A reductase according to the present invention optionally displaysactivity for reducing the carbonyl group in the side chain at C(4) ofthe isoalpha acids, converting the light-sensitive acyloin group to asecondary alcohol, and producing a light-stable isoalpha acid derivative(FIG. 1).

In another embodiment of the invention, the ketoreductase employed inthe process according to the present invention advantageously displaysminimal or no preference for catalyzing reduction of any one particularmember of the six major isoalpha acids: cis-isohumulone,trans-isohumulone, cis-isocohumulone, trans-isocohumulone,cis-isoadhumulone, and trans-isoadhumulone.

In another embodiment of the invention, the ketoreductase employed inthe process according to the present invention specifically reducescis-isohumulone, cis-isocohumulone, and cis-isoadhumulone.

In another embodiment of the invention, the ketoreductase employed inthe process according to the present invention specifically reducestrans-isohumulone, trans-isocohumulone, and trans-isoadhumulone.

In another embodiment of the invention, a mixture of 2 or moreketoreductase enzymes displaying the above substrate specificity isemployed in the process according to the present invention to reduce amixture of cis- and trans-isoalpha acids, to their respectivedihydroisoalpha acids.

In another embodiment of the invention, a mixture of 2 or moreketoreductase enzymes displaying substrate specificity can be added to areaction mixture to produce a unique mixture of dihydroisoalpha acidsthat is distinct from that produced by chemical reducing agents, such assodium borohydride.

In a further embodiment, the present invention relates to a process asdefined above, wherein the reductase enzyme is a ketoreductase.

A further embodiment of the invention relates to a process as definedabove, wherein the ketoreductase enzyme or microorganism expressing agene which encodes the ketoreductase enzyme comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ IDNO:22.

In a further embodiment, the present invention relates to a process asdefined above, wherein the ketoreductase enzyme or microorganismexpressing a gene which encodes the ketoreductase can optionally haveone or more differences at amino acid residues as compared to theketoreductase enzyme selected from SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:20, and SEQ ID NO:22.

A further embodiment of the invention relates to a ketoreductase enzymeor microorganism expressing a gene which encodes the ketoreductaseenzyme which comprises the amino acid sequence of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22.

A further embodiment of the present invention relates to a ketoreductaseenzyme or microorganism expressing a gene which encodes the reductasecan optionally have one or more differences at amino acid residues ascompared to the reductase enzyme sequence of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQID NO:19, SEQ ID NO:20, or SEQ ID NO:22.

A further embodiment of the invention relates to a ketoreductase enzymeor microorganism expressing a gene which encodes the ketoreductaseenzyme which is 99, 95, 90, 85, 80, 75 or 70 percent homologous to theketoreductase enzyme selected from SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:20 and SEQ ID NO:22.

Another aspect of the invention comprises a vector comprising apolynucleotide encoding the amino acid sequence of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22.

The invention further comprises such a vector, further comprising atleast one control sequence.

The invention further comprises a host cell comprising such a vectorcomprising a polynucleotide encoding the amino acid sequence of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22.

The invention further comprises a method for producing a ketoreductaseof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22,comprising culturing said host cell under conditions that theketoreductase is produced by said host cell.

The invention further comprises a method for producing a ketoreductaseof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22,further comprising the step of recovering the ketoreductase produced bysaid host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the enzyme catalyzed reduction of a representative epimerof isoalpha acids.

FIG. 2 shows an SDS-PAGE analysis of purified reductases.

FIG. 3 shows UPLC chromatograms for isoalpha acids incubated with (toptwo panels) and without (bottom two panels) reductase R20 for 24 hr at30° C. Peaks corresponding to the product, dihydro-(rho)-isoalpha acids,are indicated.

FIG. 4 shows a structural model of reductase R17 (dark gray, surfacerendering) with a representative substrate (trans-isohumulone, black)and cofactor (NADPH, light gray) bound to the active site cavity.

FIG. 5A shows UPLC chromatogram (A) for a ketoreductase that producesonly one diastereomer of dihydro-(rho)-isoalpha acids and FIG. 5B showsUPLC chromatogram (B) for a ketoreductase that produces bothdiastereomers of dihydro-(rho)-isoalpha acids. Peaks corresponding tothe product, dihydro-(rho)-isoalpha acids, are indicated.

FIG. 6 shows an amino acid sequence alignment, generated with ClustalOmega (www.ebi.ac.uk/Tools/msa/clustalo/) of active ketoreductasehomologs: R4, R17, R20, R21, and R23. A shared domain, containing 3regions of good (solid line) or low homology (dashed line), arehighlighted in boxes. An * (asterisk) indicates positions which have asingle, fully conserved residue. A : (colon) indicates conservationbetween groups of strongly similar properties—scoring >0.5 in the GonnetPAM 250 matrix.A . (period) indicates conservation between groups ofweakly similar properties—scoring=<0.5 in the Gonnet PAM 250 matrix.Therefore the hierchy of conservation using these symbols is *(identical)>:(colon)>. (period).

DETAILED DESCRIPTION OF THE INVENTION

In this invention, a ketoreductase enzyme replaces the function ofsodium borohydride and allows a more natural production method for thebeverage additive, dihydro-(rho)-isoalpha acids. The enzyme may be anyketoreductase specifically reducing a ketone group to a hydroxy group ofany or all isomers of isoalpha acid (co-, n- ad-, and cis/trans-). Theenzyme may be derived from, but not limited to, bacteria, fungi, orplants. The enzyme may be cofactor dependent (NADH or NADPH) orindependent.

Herein, “isoalpha acids”, “hop isoalpha acids”, and “hop-derivedisoalpha acids” may be used interchangeably.

According to the instant invention, an isoalpha acid solution issubjected to enzymatic treatment using one or more purified enzyme or amixture containing the enzyme(s) and optionally additional enzymes forcofactor recycling. The amount of enzyme depends on the incubationparameters including duration, temperature, amount and concentration ofsubstrate.

Alternatively, an isoalpha acid solution is subjected to enzymatictreatment using a mixture containing a microorganism expressing saidenzyme(s). The invention furthermore provides a process for reducingisoalpha acids according to the invention, which comprises cultivating aketoreductase-producing microorganism, if appropriate inducing theexpression of the ketoreductase. Intact cells can be harvested and addeddirectly to a reaction, in place of isolated enzyme, for the reductionof isoalpha acids as described above. Furthermore, the harvested cellscan be immobilized prior to addition to a reduction reaction. Themicroorganism can be cultivated and fermented by known methods. Themicroorganism can be bacteria or fungi.

A mixture of cis- and trans-isoalpha acids may be incubated with asingle ketoreductase displaying the capacity to reduce both isomers.Alternatively, a mixture of cis- and trans-isoalpha acids may beincubated with 2 or more ketoreductases showing varying specificitywhere the resulting product is a mixture of cis- andtrans-dihydroisoalpha acids.

Alternatively, a solution containing only cis-isoalpha acids may beincubated with ketoreductase(s) specific for the cis-isomer, and theresulting product is a solution of cis-dihydroisoalpha acids. A solutionof only cis-dihydroisoalpha acids may display advantageous bitternessand/or thermal stability properties.

Alternatively, a solution containing only trans-isoalpha acids may beincubated with ketoreductase(s) specific for the trans-isomer, and theresulting product is a solution of trans-dihydroisoalpha acids. Asolution of only trans-dihydroisoalpha acids may display advantageousbitterness properties.

Customized blends of trans- and cis-isoalphacids may be incubated with 1or more ketoreductases displaying variable substrate specificity, toproduce unique blends of dihydroisoalpha acids otherwise unattainable.

An isoalpha acid mixture may be subjected to an enzymatic reaction usingketoreductase enzyme(s) in addition to enzymes for catalyzing additionaldesired modifications, such as but not limited to, dehydrogenases,isomerases, hydratases and lyases. Enzymes of varying activity may becombined in a one pot reaction or added sequentially.

A suitable solvent to use in enzyme incubation includes water andmixtures of water with another solvent compatible with the enzyme, suchas ethanol or isopropanol. Enzymatic activity benefits from buffering ofaqueous solutions. Buffering agents include, but are not limited to:tris(hydroxymethyl)aminomethane (aka. Tris),4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (aka. HEPES), sodiumphosphate, and potassium phosphate.

The enzyme(s) and isoalpha acids are incubated within a suitable pHrange, for example pH 6 to 10, and temperature range, for example 10 to90° C., and held at this temperature for a sufficient time to convertisoalpha acids to the desired dihydro-(rho)-isoalpha acids yield.Continuous stirring will ensure a constant temperature and exposure ofsubstrate to enzyme. The reaction duration, typically 24 to 48 hours,will depend on the amount and concentration of the enzyme and substrate,solvent present, and temperature chosen.

The enzyme(s) may be free in solution, immobilized onto beads or similarmixable scaffolds, or immobilized onto a film or resin over which asolution of isoalpha acids is passed. The purity level of the enzyme mayvary from 30 to 90+% depending on the purification method.

The enzyme(s) may be removed from the final product via physicalfiltering or centrifugation. The enzyme(s) may also be rendered inactiveby extreme temperature or pH and remain in the final product.

Reductase enzymes encompassed by the present invention includeketoreductase enzymes.

Details for 23 successfully purified enzymes are listed in Table 1,including: shorthand label, sequence ID number, and amino acid sequence.

TABLE 1 Purified reductases. SEQ ID Label NO Amino Acid Sequence R1 1MSSGIHVALVTGGNKGIGLAIVRDLCRLFSGDVVLTARDVTRGQAAVQQLQAEGLSPRFHQLDIDDLQSIRALRDFLRKEYGGLDVLVNNAGIAFKVADPTPFHIQAEVTMKTNFFGTRDVCTELLPLIKPQGRVVNVSSIMSVRALKSCSPELQQKFRSETITEEELVGLMNKFVEDTKKGVHQKEGWPSSAYGVTKIGVTVLSRIHARKLSEQRKGDKILLNACCPGWVRTDMAGPKATKSPEEGAETPVYLALLPPDAEGPHGQFVSEKRVEQW R2/3 2MRLEGKVCLITGAASGIGKATTLLFAQEGATVIAGDISKENLDSLVKEAEGLPGKVDPYVLNVTDRDQIKEVVEKVVQKYGRIDVLVNNAGITRDALLVRMKEEDWDAVINVNLKGVFNVTQMVVPYMIKQRNGSIVNVSSVVGIYGNPGQTNYAASKAGVIGMTKTWAKELAGRNIRVNAVAPGFIETPMTEKLPEKARETALSRIPLGRFGKPEEVAQVILFLASDESSYVTGQVIGIDGGLVI R4 3MSVFVSGANGFIAQHIVDLLLKEDYKVIGSARSQEKAENLTEAFGNNPKFSMEVVPDISKLDAFDHVFQKHGKDIKIVLHTASPFCFDITDSERDLLIPAVNGVKGILHSIKKYAADSVERVVLTSSYAAVFDMAKENDKSLTFNEESWNPATWESCQSDPVNAYCGSKKFAEKAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDKDVKKHLNTSCELVNSLMHLSPEDKIPELFGGYIDVRDVAKAHLVAFQKRETIGQRLIVSEARFTMQDVLDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKKSKKLLGFKFRNLKETIDDTASQILKFEGRI R5 4MNQVVLVTGGSSGIGKSICLYLHEKGYIVYGTSRNPARYAHEVPFKLIALDVLDDTTITPALKTIIDAEGKLDVLVNNAGIGMLGSIEDSTAEEVKEVFETNVYGILRTCQAVLPHMRERKMGLIINVSSIAGYMGLPYRGIYSATKASVHMITEAMRMELKPYGVHACVVDPGDFATNISDNRKVAHAGRSGSVYMEEINRIEAMINAEVAHSSDPLLMGKAIEKIIRSSNPDINYLVGKPMQKLSILVRRLVPKKWFEKIIASHYNMPVK R6 5MANSGEGKVVCVTGASGYIASWLVKFLLSRGYTVKASVRDPSDPKKTQHLVSLEGAKERLHLFKADLLEQGSFDSAIDGCHGVFHTASPFFNDAKDPQAELIDPAVKGTLNVLNSCAKASSVKRVVVTSSMAAVGYNGKPRTPDVTVDETWFSDPELCEASKMWYVLSKTLAEDAAWKLAKEKGLDIVTINPAMVIGPLLQPTLNTSAAAILNLINGAKTFPNLSFGWVNVKDVANAHIQAFEVPSANGRYCLVERVVHHSEIVNILRELYPNLPLPERCVDENPYVPTYQVSKDKTRSLGIDYIPLKVSIKETVESLKEKGFAQF R7 6MTLSSAPILITGASQRVGLHCALRLLEHGHRVIISYRTEHASVTELRQAGAVALYGDFSCETGIMAFIDLLKTQTSSLRAVVHNASEWLAETPGEEADNFTRMFSVHMLAPYLINLHCEPLLTASEVADIVHISDDVTRKGSSKHIAYCATKAGLESLTLSFAARFAPLVKVNGIAPALLMFQPKDDAAYRANALAKSALGIEPGAEVIYQSLRYLLDSTYVTGTTLTVNGGRHVK R8 7MSLQGKVALVTGASRGIGQAIALELGRQGATVIGTATSASGAERIAATLKEHGITGTGMELNVTSAESVEAVLAAIGEQFGAPAILVNNAGITRDNLMLRMKDDEWFDVIDTNLNSLYRLSKGVLRGMTKARWGRIISIGSVVGAMGNAGQANYAAAKAGLEGFSRALAREVGSRGITVNSVTPGFIDTDMTRELPEAQREALQTQIPLGRLGQADEIAKVVSFLASDGAAYVTGATVPVNGGMYM R9 8MDLTNKVVVVTGGSAGLGEQICYEAAKQGAVVVVCARRINLIGKVREQCAVLSGREAFSYQLDIADPESVERVVEAISAEVGPIDVLVNNAGFGLFENFVEIDLAVARQMFDVNVLGMMTFTQKVAIKMIEAGQGHIINVASMAGKMATAKSTVYSATKFAVLGFSNALRLELKPLGVAVTTVNPGPIQTEFFDKADPTGTYLAAVDKIVLDPTKLAKEVVGSMGTSRREINRPFVMEAAARFYTLFPHLGDFIAGNILNKK R10 9MRRILITGANGFVGQILCSMLRQAGHHVIALVGAESALSSHADESVRCDIRDASGLEQALCRAAPTHVVHLAAITHVPTSFNNPVLTWQTNVMGSVNLLQALQRSAPEAFVLFVSSSEVYGETFKQGTALGEDSACKPMNPYAASKLAAEAAFNEYFRQGRKGIVVRPFNHIGARQSPDFATASFARQIALIEAGKQAPQLKVGNLQAARDFLDVHDVCDAYVALLQLADEQERYPGCLNICRGEPTSLQTLLTQLMALSSSVIEVTIDPDRMRPSDIPSAFGNNSAMRCATGWKPKTKLDDTLEALLNYWRHEVISAV R11 10MSLLLEPYTLRQLTLRNRIAVSPMCQYSSVDGLANDWHLVHLGSRAVGGAGLVISEAMAVTPDGRITPEDLGLWNDEQIEPLQRITRFINTQGAVAGIQLAHAGRKASTWRPWLGKHGSVPLTEGGWTPVGPSAIAFDPQHTAPLQLSETQIQELIKAFVDSARRALTAGFKVVEIHAAHGYLLHQFLSPLSNQRTDQYGGSFENRIRLTLQVTEAVRAVWPQELPLFVRVSATDWVEDGWNAEETVELARRLKALGTDLIDVSSGGTSANAEIPVGPGYQTRFAEQVRKEADIATGTVGMITDPAQAEHILRTGQADIILLARELLRDPYWPLRADEDLGGRQATWPAQYQRATHRDQPIHESDLRD R12 11MSSSSLRVLAIGNNPNILFYTSRFQLAKNIDLYHVNDSKSCQFEIETEYYGKDRFELENHFTSIEHLTEALSSKSSEAVFDIIIMSAPSLQELSSLASKLTSIIDSNTKIFLESSGFIQLEPFVKLSMESPHVNVFSILTDLDIRQIGPNHFKHFPSTAKENTIYLGESKSSTEKYSSGVITLLTTFEKLFAKLFSNIKINLCNFSSIEFLSQQWKLAISRICFDPLLIMFEQENPSDLDQQIIAKPLISGLVTEIITVAKTMGARLNSSHDNENSLLSLWKNSYHSTNKPPALVYHFIHQTTPLNIDILLLQTILLADDFGIKTPYLEFLYSVLSQFERL NSGR13 12 MEYRKVGKWGVKISELSLGSWLTFGKQLDLDTATEVVKKAFNSGINFFDTAEAYAGGIAEAMLGKILKNFRREDLVVSTKIFWGGSGPNDLGLSKKHLLEGTWNSLKRLQMDYVDILYCHRPDPNVPMEEVVFAMDYILREGLALYWGTSEWSAKEIEEAHRVCKELGVMPPIVEQPQYNMFVRERVEKEYAPLYEKYGMGLTTYSPLASGLLSGKYNNGIPEGSRLATFPQVRKWLEEGGLLNEKTFKKLRKLQNIADQLGASLPQLAIAWILKNKNVSSVILGVSRPEQLEENLKAVEIKEKLTEDVMEEIEKILNE R14 13MTLANLPPLVTVFGGSGFVGRHVVRMLAKRGYRIRVAVRRPDLAGFLQPLGNVGQISFAQANLRYRDSIIKAVEDADHVVNCVGILAESGRNTFDAVQEFGAKAIAEAARDTGATLTHISAIGADANSQTGYGRTKGRAEAAIHSVLPGAVILRPSIIFGPEDDFFNKFAKMARNLPFLPLIGGGKTKFQPVYVEDVAEAVARSVDGKLKPGAIYELGGPDVMTFRDCLEAVLAATYRERSFVNLPFGVASMIGKLASLVPLITPPLTPDQVTMLKKDNVVSAEAEKKGLTLEGIGITPVRVASVLPSYMVQYRQHGQFSNAGKAA R15 14MTAEVFDPRALRDAFGAFATGVTVVTASDAAGKPIGFTANSFTSVSLDPPLLLVCLAKSSRNYESMTSAGRFAINVLSETQKDVSNTFARPVEDRFAAVDWRLGRDGCPIFSDVAAWFECSMQDIIEAGDHVIIIGRVTAFENSGLNGLGYARGGYFTPRLAGKAVSAAVEGEIRLGAVLEQQGAVFLAGNETLSLPNCTVEGGDPARTLAAYLEQLTGLNVTIGFLYSVYEDKSDGRQNIVYHALASDGAPRQGRFLRPAELAAAKFSSSATADIINRFVLESSIGNFGIYFGDETGGTVHPIANKDAHS R16 15MDEVILVTGAAKGIGLATVKRLSSQGARVILNVHHEIEATDWQALTAEYPRLTQLVGDVSDDQSAANLIDTVMTNFGRLDGLVNNAGVTHDQLLTRLHAEDFMSVIQTNLLGTFNMTKYALKVMQRQRQGAIVNVASVVGLHGNVGQANYAASKAGIIGLTKTTAKEAARRQVRCNAVAPGMITTAMTAQLNDRVTAAALSDIPLKRFGTPDEIAQAIDFLLHQPYLTGQVLTVDGGMTI R17 16MRVLLTGGSGFIAAHILDILLSRGHTVITTVRSQQKIDAIKAAHPDVPASKLDFFIVEDIAKENAFDECLKKFGEGLEAVLHTASPFHFNVTDTKKDLLDPAIIGTTAILHAIKKFAPSVTRVVVTSSFASIIDASKGNWPDHTYTEEDWNPITLSEAVENPSNGYRASKTFAEKAAWEFVEKENPNFTLSTMNPPLVLGPIVHYLNSLDALNTSNQRVRDVLQGKWKEEIPGTGTFIWIDVRDLALAHVKAIEIAEAAGKRFFITEGYFSNKEICEIIRKNFPEDGGELPGKEVKGGGYPEGGIYKFDNARTRSVLGLEFRGLEESIVDLVKSLKEVGV R18 17MSRNLALVTGSTQGIGLAVAKELAIKHNYQVLLGVRNTKAGEEIASDLRKEGHEASVVELDLTSADSIDKAVKHIDEKYGYLDVLINNAGVLLDRQEGLSTWDLFSKTFTTNVFGTGCLTQSLLPLLRKAKNSPPRIVFVTSVMGSLTKATDETTTYYNIDYKAYDASKAAVNMLMFNFARELDAVGGKVNSVCPGLVKTGLTNYHEWGTSPETGAERIVEMATIGEDGPTKTISDRNGELPL R19 18MDLQNKRVLVTGSTQGIGAATALAFAQKGCQVLLNGRRPELPEEIADQLEKIGADYQYFSADVSDEGAIKQLFKEIGEIDILVNNAGITKDQIMIGMKLADFDQVIKVNLRSSFMLTQKALKKMLKKRSGAIINMASIVGQHGNAGQANYAASKAGVIALTQTAAKEAAGRGVRVNAIAPGMIASQMTAVLPDEVKEQALSQIPLARFGKAEEVAQAAVFLAENDYVTGQTLVVDGGMTI R20 19MTKVLVAGGSGFIGAHILEQLLAKGHSVVTTVRSKEKAQKILDAHKAEADRLEVAIVPEIAREDAFDEVVKTPGIEVVIHPASPCHLNFTDPQKELIDPAVLGTTNILRAIKRDAPQVRRVIITSSVAAIFNTKDPVSTLTEQSWNPNDLSNIHDSRAVAYCVSKTLAERAAWDYVDQEKPNFDLVTVNPPLVLGPVVGHFSNVDSINASNECLANLVRGKWRDEIPPTGPVNIWIDVRDVAAAHVRAMERQEAGGKRLFTVGGRFSYTKIAEIVREHGPDRFKDKMPRAEARSGDANYTGPVLKFDNGETNRILGIEWTPLEKSVLDFVESIKEFDL R21 20MTKVLLTGGSGFIAAHILEQLLAKNYTVITTVRTKSKADLIKEAHADLVKSGRLSVAIVPDIAVLSAFDDLVAKIASGPDGDLEYVVHTASPLFFTFTDAQKEIITPALNGTRGILEAVKRSAPKVKRVVITSSFAAILSEDDFTNPNATFSESSWNPDTVKDANRSIATGYHVSKVESERLAWDFIKNEKPNFDLVTVNPPLVLGPVAHSLASVDAINASNERIADLLRGKWKAEIPETGAVDLYIDVRDTAKAHIKALELPEASGHRLFPVASRTSNHEIAKIIRDNFPEFAERLPGPEVKGGEHVDENKAYKWNCDETNKLLKIDWIPIEQSMIDTVNSLKDKGI R22 21MPTVSPGSKVLVTGANGFIAIWVVRRLLEEGYSVRGTVRAASKASHLKDIFKSYGEKLEVVVVPDFTKEGAFDELIKGMDAIQHIASPGPANTDDLYEIVNPAVDGTLNLLNTALKHGSGLKRIVITSGAGAIIDTTTAWKFYNDHKNVIKWDLTVLNPVFVFGPPIHEIGASPMTLNSSMVHFWVNVISTDTPKTKEGLSFAASWVDVRDVAQGHVLALQKEAAGGERIILSEGSFVWQDWVDVANKFKSKRELPKGMPEIERVYKFQMDASKATRILGITYRSKEDTMKDLLEDFERRGW R23 22MKVLLTGGSGFIATHCLDALLKHGHEVVITVRSAEKGQALVDLFKGQKVSYTIVKDISVPGAFDQAVISDPPFDAVVHTASPFHYDVQDNKRDLLDPAIIGTTGILESIQKGAPSVKKVVVTSSFAAISNPTAPPKVYDETVWNQMTMEEALTTKDPQAVYRGSKTFAEKAAWEFVEREKPNFTLTVLNPPVSHFLFSRHKDVAVTFFSDSFQHCRWSTARSCTPWHHWTISTPRASES

Almost all candidates were sufficiently pure (>80% protein content isthe protein of interest) after one-step purification (See FIG. 2).

Reductase enzymes encompassed by the present invention include thosecomprising the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQID NO:19, SEQ ID NO:20, and SEQ ID NO:22.

Reductase enzymes encompassed by the present invention also includethose having one or more differences at amino acid residues as comparedto the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:22.

Reductase enzymes encompassed by the present invention also includethose comprising an amino add sequence which is identical by at least40% (including at least 50%, at least 60%, at least 70%, at least 80%,al least 85%, at least 90%, and at least 95%) to the following aminoacid sequences: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ IDNO:20, and SEQ ID NO:22.

As used herein, “percentage of sequence homology,” “percent homology,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence homology is performed using the BLAST and BLAST 2.0 algorithms(See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul et al., Nucleic Acids Res. 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Promiscuous enzymes may catalyze the same chemical reaction despitepossessing low shared amino acid identity. Ketoreductase R4 (SEQ IDNO:3) was initially selected for screening due to its promiscuous nature[Guo et al. Biochim. Biophys. Acta 2014, 1844]. Five additionalketoreductases (R17 (SEQ ID NO:16), R20 (SEQ ID NO:19), R21 (SEQ IDNO:20), R22 (SEQ ID NO:21) and R23 (SEQ ID NO:22)) that contain the sameenzyme domain (IPR001509: NAD-dependent epimerase/dehydratase) and shareamino acid identity to R4 (SEQ ID NO:3) were acquired as syntheticgenes, purified and characterized. Reductases were purposely selected atincreasingly lower sequence identity in order to establish a sequenceidentity cutoff.

Despite sharing a relatively low percent identity (34-39% over entirelength of the enzyme) to R4 (SEQ ID NO:3), enzymes R17 (SEQ ID NO:16),R20 (SEQ ID NO:19), R21 (SEQ ID NO:20) and R23 (SEQ ID NO:22) catalyzethe transformation of isoalpha acids to dihydro-(rho)-isoalpha acids .R22 (SEQ ID NO:21) which shares 33% identity to R4 (SEQ ID NO:3) doesnot catalyze the transformation of isoalpha acids todihydro-(rho)-isoalpha acids but is otherwise an active enzyme aspurified (established by measuring enzyme-catalyzed oxidation activityof isopropanol).

A feature that separates functional from nonfunctional reductases forobtaining dihydro-(rho)-isoalpha acids is illustrated by a multiplesequence alignment (FIG. 6). Ketoreductase R4 (SEQ ID NO:3), and all R4(SEQ ID NO:3) homologs characterized as capable of converting isoalphaacids to dihydro-(rho)-isoalpha acids herein, possess a domain occurringbetween residues 100 and 200, composed of 13 amino acids of goodhomology (>53% identity) and 9 amino acids of high homology (>55%)separated by 36-39 amino acids of low homology (38-46%). This domain ismissing in the nonfunctional R22 polypeptide (SEQ ID NO:21). The domainis thus deemed a hallmark of ketoreductase activity for obtainingdihydro-(rho)-isoalpha acids.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others.

The term “effective amount” refers to that quantity of a reductase whichis sufficient to transform isoalpha acids into dihydro-(rho)-isoalphaacids. Determination of an effective amount for a given administrationis well within the ordinary skill in the pharmaceutical arts.

In a method for preparing dihydro-(rho)-isoalpha acids, an isoalpha acidsolution is subjected to enzymatic treatment using one or more purifiedreductase enzymes or a mixture containing a reductase enzyme(s) andoptionally additional enzymes for cofactor recycling, in an amounteffective to transform the isoalpha acids into dihydro-(rho)-isoalphaacids. The amount of enzyme depends on the incubation parametersincluding duration, temperature, amount and concentration of substrate.

Alternatively, an isoalpha acid solution is subjected to enzymatictreatment using a mixture containing a microorganism expressing saidenzyme.

A mixture of cis- and trans-isoalpha acids may be incubated with asingle reductase/ketoreductase displaying the capacity to reduce bothisomers. Alternatively, a mixture of cis- and trans-isoalpha acids maybe incubated with 2 or more ketoreductases showing varying specificitywhere the resulting product is a mixture of cis- andtrans-dihydroisoalpha acids.

Customized blends of trans- and cis-isoalphacids may be incubated with 1or more reductases/ketoreductases displaying variable substratespecificity, to produce unique blends of dihydroisoalpha acids otherwiseunattainable.

An isoalpha acid mixture may be subjected to an enzymatic reaction usinga reductase enzyme in addition to enzymes for catalyzing additionaldesired modifications, such as but not limited to, dehydrogenases,isomerases, hydratases and lyases. Enzymes of varying activity may becombined in a one pot reaction or added sequentially.

A suitable solvent to use in the enzyme incubation includes water andmixtures of water with another solvent compatible with the enzyme, suchas ethanol or isopropanol. Enzymatic activity benefits from buffering ofaqueous solutions. Buffering agents include, but are not limited to:tris(hydroxymethyl)aminomethane (aka. Tris),4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (aka. HEPES), sodiumphosphate, and potassium phosphate.

The enzyme and isoalpha acids are incubated within a suitable pH range,for example pH 6 to 10, and temperature range, for example 10 to 90° C.,and held at this temperature for a sufficient time to convert isoalphaacids to the desired dihydro-(rho)-isoalpha acids yield. Continuousstirring will ensure a constant temperature and exposure of substrate toenzyme. The reaction duration, typically 24 to 48 hours, will depend onthe amount and concentration of the enzyme and substrate, solventpresent, and temperature chosen.

The reductase enzyme may be free in solution, immobilized onto beads orsimilar mixable scaffolds, or immobilized onto a film or resin overwhich a solution of isoalpha acids is passed. The purity level of theenzyme may vary from 30 to 90+% depending on the purification method.

The reductase may be removed from the final product via physicalfiltering or centrifugation. The enzyme may also be rendered inactive byextreme temperature or pH and remain in the final product.

The present invention is a novel method of utilizing reductases totransform isoalpha acids into dihydro-(rho)-isoalpha acids. Codonoptimized reductase genes have achieved yields of upwards of 100 mgpurified enzyme per L cell culture in E. coli BL21(DE3). All enzymeswere characterized with NADPH as the cofactor. The reductasescharacterized in this study possess an enzymatic activity that has notbeen described previously. These enzymes form a basis for the novelbiocatalysts which may be utilized in a novel biotransformation toreplace current processes utilizing sodium borohydride.

EXAMPLES

The following examples illustrate the invention without limiting itsscope.

Example 1—Reductase Preparation and Screening Methods CandidateIdentification

Reductase candidates were selected after an extensive search of theliterature for characterized enzymatic reactions of a similar nature tothe desired reaction, followed by bioinformatic mining of three publicprotein sequence databases: UniProt (www.uniprot.org/), Pfam(//pfam.xfam.org/), and InterPro (www.ebi.ac.uk/interpro/E. coli).Bioinformatics relied on BLASTP sequence alignments(//blast.ncbi.nlm.nih.gov/Blast.cgi) between characterized enzymes andreductase candidates.

Enzyme Expression and Purification

Plasmid DNA was acquired in several manners: 1) in an expression vectorfrom the DNASU Plasmid Repository (www.dnasu.org), 2) in a cloningvector from DNASU Plasmid Repository and subsequently cloned into anin-house expression vector, 3) as a synthetic gene in an expressionvector from Atum (www.atum.bio), or 4) as a synthetic gene in anexpression vector from General Biosystems (www.generalbiosystems.com).Synthetic genes were codon-optimized for expression in E. coli.

5 mL Luria Broth with appropriate antibiotics was inoculated from anagar plate of E. coli BL21 (DE3) containing the expression vector ofinterest and incubated at 30° C. with shaking overnight. The followingday, the overnight culture was back-diluted 1:100 into fresh 0.5 L LuriaBroth with antibiotics and incubated at 37° C. for 2-3 hr with 220 rpmshaking until an optical density of 0.5 was reached. Cultures wereinduced with 0.2 mM final concentration of isopropylβ-D-1-thiogalactopyranoside (IPTG) and incubated at 25° C. with 180 rpmshaking for 16 h. Cells were harvested by centrifugation at 4800 rpm for15 min. The cell pellet was resuspended in 12 mL of Bind Buffer (10 mMHEPES, 50 mM NaCl, pH 7.5) and cells were lysed via sonication for 15min (5 sec on, 5 sec off). Cell lysate was clarified via centrifugationat 10,000 rpm for 20 min. Tagged protein was purified from clarifiedcell lysate via cobalt affinity, maltose affinity, or glutathioneaffinity chromatography. Protein solutions were exchanged into ProteinStorage Buffer (20 mM Tris-HCl, 50 mM NaCl pH 7.0) via centrifugalfiltration. Protein concentration was measured via absorbance at 280 nmusing extinction coefficients calculated by using the appropriate aminoacid sequence. Glycerol added to a final concentration of 20% and enzymesolutions frozen at −20 or −80° C.

Isoalpha Acids Reduction Assay

Purified enzyme candidates were tested for their ability to reduceisoalpha acids. The specific reaction entails reducing a specific ketonegroup to a hydroxy group of any or all isomers and congeners of isoalphaacid (co-, n- ad-, and cis/trans-). In a 2 mL microcentrifuge tube, 100uL of enzyme solution (final concentration of 0.15-1.8 mg/mL enzyme) wasadded to 900 uL of buffered aqueous solution with cofactor recycling byglucose dehydrogenase (263 mM sodium phosphate, 1.7 mM magnesiumsulfate, 1.1 mM NADP+, 1.1 mM NAD+, 80 mM D-glucose, 4.3 U/mL glucosedehydrogenase, pH 7.0). 5 uL of alkaline isoalpha acid solution(ISOLONE®, 29% isoalpha acids) was added for a final concentration of0.29% isoalpha acids. The reaction was incubated at 30° C. with orbitalshaking at 180 rpm for 24 hours. The obtained reaction mixture wasfiltered to remove enzyme. Isoalpha acids and dihydro-(rho)-isoalphaacids were detected by UPLC-MS/MS. A negative control sample containsall the above reaction components where the enzyme solution was replacedwith Protein Storage Buffer.

Results Candidate Selection

Based on public functional annotations and amino acid sequencesimilarity, 60 unique enzyme sequences were identified as beingreductase candidates.

Enzyme Expression and Purification

30 candidates were selected for expression and purification based on theavailability of DNA and sufficient sampling of the diversity of aminoacid sequences represented in the initial group of 60 candidates. Mostcandidates displayed good expression and solubility levels in the E.coli BL21(DE3) with yields varying from 5 to 100 mg purified protein perliter culture. Several candidates were abandoned due to poor solubilityin the host organism.

Reductase Characterization

Enzymes were determined to reduce isoalpha acids if peaks correspondingto cis/trans- co/ad/n-dihydro-(rho)-isoalpha acid were detected via UPLCat a greater intensity than a control sample lacking enzyme. Ten uniqueenzymes were determined to be isoalpha acid reductases (See FIG. 3).Details of these enzymes are summarized in Table 2. Due to solubilityand enzyme yield, the final concentration of in-house enzymes in theassay varied from 0.15-1.8 mg/mL. Lower enzyme concentration contributesto the dihydro-(rho)-isoalpha acids yield.

Enzymes were initially tested for reductase activity in the presence ofglucose, glucose dehydrogenase, and NAD in order to recycle the NADPrequired for isoalpha acid reduction. After determination of reductaseactivity, enzymes were characterized for their ability to oxidizeisopropanol, a more economical alternative for cofactor recycling.Ability to efficiently oxidize isopropanol is indicated in Table 2.

TABLE 2 Novel isoalpha acid reductases characterized. Label IsoalphaAcid Reduction Isopropanol Oxidation R2 Yes No R4 Yes Yes R7 Yes No R9Yes Yes R13 Yes No R14 Yes Yes R17 Yes Yes R20 Yes Yes R21 Yes NotTested R23 Yes Not Tested

Substrate Specificity

The ideal ketoreductase for biotransformation purposes shows nosubstrate specificity for the isohumulone congeners which vary based onside chain composition (conferring n-, ad-, and co-isohumulone).Additionally, the ketoreductase shows no specificity for the isohumulonecis and trans isomers which vary spatially at the C4 tertiary alcoholgroup proximal to the site of enzymatic reduction. Substrate specificityis dictated by the amino acid sequence and thus the geometry of thesubstrate binding pocket of an enzyme. Larger binding pocketsaccommodate larger substrates, as well as a greater variety ofsubstrates, compared to more restricted binding pockets. (See FIG. 4).

Two varieties of reduction stereospecificities were observed among thecharacterized reductases (See FIG. 5).

Despite the presence of two additional ketone groups on the isoalphaacid molecule, only the desired reduction at the C4 side chain wasobserved for all characterized ketoreductases.

Example 2—Enzyme Treatment of Hop Derived Isoalpha Acids with CofactorRecycling by Isopropanol Oxidation

In a 1.5 mL microcentrifuge tube, 10 mg reductase is resuspended in 700uL of buffered aqueous solution (eg. Sodium Phosphate pH 7.5). 290 uL ofisopropanol is added. 10 uL of alkaline isoalpha acid solution (29%isoalpha acids) is added for a final concentration of 0.29% isoalphaacids. The reaction is incubated at 30° C. with orbital shaking at 180rpm for 48 hours. The obtained reaction mixture is filtered to removeenzyme. Isoalpha acids and dihydro-(rho)-isoalpha acids are quantifiedby HPLC.

Example 3—Enzyme Treatment of Acidified Hop Derived Isoalpha Acids withCofactor Recycling by Isopropanol Oxidation

Isoalpha acids are treated in a manner described in Example 2, where thesource of isoalpha acids is a highly concentrated material (68.9%isoalpha acids) having a pH<7.

Example 4—Enzyme Treatment of Hop Derived Isoalpha Acids with CofactorRecycling by Glucose Dehydrogenase

Isoalpha acids are treated in a manner described in Example 2, with theexception that isopropanol is replaced with 4.3 U/mL GlucoseDehydrogenase, 0.7 g/L mM NAD, and 14.4 g/L D-glucose.

Example 5—Enzyme Treatment of Hop Derived Isoalpha Acids withoutCofactor Recycling

Isoalpha acids are treated in a manner described in Example 2, with theexception that isopropanol is replaced with an equimolar amount of NADPHas substrate.

Example 6—Enzyme Treatment of Hop Derived Isoalpha Acids withThermostable Reductase

Naturally thermostable reductases are obtained from thermophilicbacterial and archaeal organisms, such as Thermotoga maritima. In a 1.5mL microcentrifuge tube, 100 uL enzyme solution (1.5-15.0 mg/mL enzyme)is added to 900 uL of buffered aqueous solution (263 mM Sodium PhosphatepH 7.0, 1.7 mM magnesium sulfate, 4.3 U/mL Glucose Dehydrogenase, 1.1 mMNADP+, 1.1 mM NAD+, 80 mM D-glucose). ISOLONE® Isomerized Hop Extractsolution (29% isoalpha acids) is added for a final concentration of0.145-16% isoalpha acids. The reaction is incubated at 60-80° C. withorbital shaking at 180 rpm for 24 hours. The obtained reaction mixtureis filtered to remove enzyme.

Example 7—Enzyme Treatment of Hop Derived Isoalpha Acids with CofactorRecycling by Ethanol Oxidation

Isoalpha acids are treated in a manner described in Example 2, with theexception that isopropanol is replaced with ethanol.

Example 8—Enzyme Treatment of Hop Derived Isoalpha Acids withImmobilized Ketoreductase Via SiO₂

A ketoreductase is adsorbed on SiO₂ and crosslinked with glutaraldehydeto yield an immobilized ketoreductase material. Isoalpha acids aretreated with the immobilized ketoreductase in a manner described inExample 2. The obtained reaction mixture is centrifuged at 10,000 g toremove immobilized enzyme.

Example 9—Enzyme Treatment of Hop Derived Isoalpha Acids withImmobilized Ketoreductase Via DEAE-Cellulose

A ketoreductase is crosslinked with glutaraldehyde and adsorbed ontoDEAE-cellulose to yield an immobilized ketoreductase material. Isoalphaacids are treated with the immobilized ketoreductase in a mannerdescribed in Example 2. The obtained reaction mixture is centrifuged at10,000 g to remove immobilized enzyme.

Example 10—Enzyme Treatment of Hop Derived Isoalpha Acids withImmobilized Ketoreductase Via PEI-Treated Alumina

A ketoreductase is crosslinked with glutaraldehyde and adsorbed ontopolyethylimine (PEI)-treated alumina to yield an immobilizedketoreductase material. Isoalpha acids are treated with the immobilizedketoreductase in a manner described in Example 2. The obtained reactionmixture is centrifuged at 10,000 g to remove immobilized enzyme.

Example 11—Enzyme Treatment of Hop Derived Isoalpha Acids withCo-Immobilized Enzymes

A reductase and cofactor recycling enzyme, such as glucosedehydrogenase, are immobilized sequentially or together in a singlecomposition utilizing any of the above-mentioned methods to yield acoimmobilized material. Coimmobilized material is added to aconcentration of 0.1-10 mg/mL in buffered aqueous solution (50-250 mMsodium phosphate, 0.1-1.0 mM NADPH, 10-40% isopropanol, pH 7-9).ISOLONE® Isomerized Hop Extract solution (29% isoalpha acids) is addedfor a final concentration of 0.145-16% isoalpha acids. The reaction isincubated at 30-40° C. with orbital shaking at 180 rpm for 24 hours. Theobtained reaction mixture is centrifuged at 10,000 g to removeimmobilized enzyme.

Example 12 —Enzyme Treatment of Hop Derived Isoalpha Acids withCrosslinked/Spheronized Cells

A cell (bacterial, fungal, plant) expressing the reductase iscrosslinked with polyamine/glutaraldehyde, extruded and spheronized toyield an immobilized reductase material. Immobilized reductase is addedto a concentration of 0.1-10 mg/mL in buffered aqueous solution (50-250mM sodium phosphate, 0.1-1.0 mM NADPH, 10-40% isopropanol, pH 7-9).ISOLONE® Isomerized Hop Extract solution (29% isoalpha acids) is addedfor a final concentration of 0.145-16% isoalpha acids. The reaction isincubated at 30-40° C. with orbital shaking at 180 rpm for 24 hours. Theobtained reaction mixture is centrifuged at 10,000 g to removeimmobilized enzyme.

Example 13—Enzyme Treatment of Hop Derived Isoalpha Acids withCrosslinked/Entrapped Cells

A cell (bacterial, fungal, plant) expressing the reductase iscrosslinked with glutaraldehyde and entrapped within gelatin or polymerbeads to yield an immobilized reductase material. Immobilized reductaseis added to a concentration of 0.1-10 mg/mL in buffered aqueous solution(50-250 mM sodium phosphate, 0.1-1.0 mM NADPH, 10-40% isopropanol, pH7-9). ISOLONE® Isomerized Hop Extract solution (29% isoalpha acids) isadded for a final concentration of 0.145-16% isoalpha acids. Thereaction is incubated at 30-40° C. with orbital shaking at 180 rpm for24 hours. The obtained reaction mixture is centrifuged at 10,000 g toremove immobilized enzyme.

Example 14—Enzyme Treatment of Hop Derived Isoalpha Acids with LivingCells

A microorganism (bacteria, fungus) expressing the reductase is grown viafermentation to high density, harvested, washed, and pelleted to formcell paste. Cell paste is resuspended in fresh growth media containing0.145-16% isoalpha acids. The cell culture is incubated at 25-37° C.with mixing for 24-72 hours. The cell culture is centrifuged at 10,000 gto remove cells from spent growth media. Dihydro-(rho)-isoalpha acidsare extracted from the spent growth media with ethanol.

Example 15—Enzyme Treatment of Hop Derived Isoalpha Acids with CellLysate

A microorganism (bacteria, fungus) expressing the reductase is grown viafermentation to high density, harvested, washed, and lysed to yield acrude cell lysate. Isoalpha acids are added to the crude cell lysate toa final concentration of 0.145-16% isoalpha acids. The cell culture isincubated at 25-40° C. with mixing for 24 hours. The reaction mixture iscentrifuged at 10,000 g or filtered to remove cellular material from thelysate. Dihydro-(rho)-isoalpha acids are extracted from the clarifiedlysate with ethanol.

Example 16—Enzyme Treatment of Hop Derived Isoalpha Acids withPsychrophilic Reductase

Enzyme treatment where the reductase is a homolog from a psychrophilic(cold tolerant) microorganism. Reductase is added to a concentration of0.1-10 mg/mL in buffered aqueous solution (50-250 mM sodium phosphate,0.1-1.0 mM NADPH, 10-40% isopropanol, pH 7-9). ISOLONE® Isomerized HopExtract solution (29% isoalpha acids) is added for a final concentrationof 0.145-16% isoalpha acids. The reaction is incubated at 0-20° C. withorbital shaking at 180 rpm for 24 hours. The obtained reaction mixtureis filtered to remove enzyme.

Example 17—Enzyme Treatment of Hop Derived Isoalpha Acids with NADHCofactor Recycling

Enzyme treatment where the NADPH cofactor is substituted with NADH.Isoalpha Acids are treated in a manner described in Example 2 but theNADP is replaced with NAD.

Example 18—Enzyme Treatment of Hop Derived Isoalpha Acids with CofactorRecycling Via Ethanol Oxidation

Enzyme treatment where the isopropanol starting material is substitutedwith ethanol, wherein a reductase is added to a concentration of 0.1-10mg/mL in buffered aqueous solution (50-250 mM sodium phosphate, 0.1-1.0mM NADH, 10-40% ethanol, pH 7-9). ISOLONE® Isomerized Hop Extractsolution (29% isoalpha acids) is added for a final concentration of0.145-16% isoalpha acids. The reaction is incubated at 30-40° C. withorbital shaking at 180 rpm for 24 hours. The obtained reaction mixtureis filtered to remove enzyme.

Example 19—Enzyme Treatment of Hop Derived Isoalpha Acids Followed byExtraction

Enzyme treatment followed by extraction to increase final concentrationof dihydro-(rho)-isoalpha acids. Isoalpha acids are treated in a mannerdescribed in Example 2. The obtained reaction mixture is filtered toremove enzyme and extracted with food-grade solvent to achieve a desiredconcentration of dihydro-(rho)-isoalpha acids.

Example 20—Enzyme Treatment of Hop Derived Isoalpha Acids Followed byThermal Inactivation

Isoalpha acids are treated in a manner described in Example 2. Thereaction is incubated at 30-40° C. with orbital shaking at 180 rpm for24 hours. The obtained reaction mixture is heated at 80-100° C. for10-30 minutes to inactivate enzyme.

Example 21—Enzyme Treatment of Hop Derived Isoalpha Acids Followed byChemical Inactivation

Isoalpha acids are treated in a manner described in Example 2. Thereaction is incubated at 30-40° C. with orbital shaking at 180 rpm for24 hours. Food-grade ethanol is added to a final concentration of >50%to inactivate enzyme.

Example 22—Enzyme Treatment of Hop Derived Isoalpha Acids withImmobilized Ketoreductase Recycling

A ketoreductase is crosslinked with glutaraldehyde and absorbed ontoDEAE-cellulose to yield an immobilized ketoreductase material. Isoalphaacids are then treated with the immobilized ketoreductase in a mannerdescribed in Example 2. The obtained reaction mixture is centrifuged at10,000 g to separate immobilized ketoreductase from the reactionsolution. Immobilized ketoreductase is recovered, washed with water oraqueous buffer, and re-used in a new reaction mixture.

CONCLUSIONS

23 ketoreductases have been characterized as transforming isoalpha acidsinto dihydro-(rho)-isoalpha acids. The ketoreductases characterized inthis study possess an enzymatic activity that has not been describedpreviously. The ketoreductases characterized in this study all reduce aketone group into an alcohol and are thus ketoreductases. These resultsdemonstrate that a ketoreductase biocatalyst may be employed to convertisoalpha acids to dihydro-(rho)-isoalpha acids in a novelbiotransformation process. The present invention replaces currentchemical processes utilizing sodium borohydride.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

CITED REFERENCES

-   -   1. Sodium Borohydride; MSDS No. S9125; Sigma-Aldrich Co.: Saint        Louis, Mo. Nov. 1, 2015. (accessed Jun. 8, 2017).    -   2. Robinson, P. K., Enzymes: principles and biotechnological        applications. Essays Biochem 2015, 59, 1-41.    -   3. Hult, K.; Berglund, P., Enzyme promiscuity: mechanism and        applications. Trends Biotechnol. 2007, 25 (5), 231-238.    -   4. Nobeli, I.; Favia, A. D.; Thornton, J. M., Protein        promiscuity and its implications for biotechnology. Nat.        Biotechnol. 2009, 27 (2), 157-167.    -   5. Pozen, M., Enzymes in Brewing. Ind. Eng. Chem, 1934, 26 (11),        1127-1133.    -   6. Praet, T.; Opstaele, F.; Jaskula-Goiris, B.; Aerts, G.; De        Cooman, L., Biotransformations of hop-derived aroma compounds by        Saccharomyces cerevisiae upon fermentation. Cerevisia, 2012, 36,        125-132.    -   7. Wallerstein, L. (1947) Bentonite and Proteolytic Enzyme        Treatment of Beer, U.S. Pat. No. 2,433,411.    -   8. Ghionno, L.; Marconi, O; Sileoni, V.; De Francesco, G.;        Perretti, G., Brewing with prolyl endopeptidase from Aspergillus        niger the impact of enzymatic treatment on gluten levels,        quality attributes, and sensory profile. Int. J. Food Sci.        Technol, 2017, 52 (6), 1367-1374.    -   9. Gros, J.; Tran, T. T. H.; Collin, S., Enzymatic release of        odourant polyfunctional thiols from cysteine conjugates in        hop. J. Inst. Brew. 2013, 119 (4), 221-227.

1. A process for the preparation of dihydro-(rho)-isoalpha acids,comprising treating isoalpha acids with a ketoreductase enzyme or amicroorganism expressing a gene that encodes the ketoreductase.
 2. Theprocess according to claim 1, wherein the process is carried out in anaqueous system.
 3. The process according to claim 2, wherein the processis carried out under mild temperature and pH conditions.
 4. The processaccording to claim 1, comprising adding the ketoreductase enzyme andNADPH or NADP to a mixture of isoalpha acids followed by incubation. 5.The process according to claim 1, comprising adding the ketoreductaseenzyme and NADPH or NADP to a mixture of isoalpha acids in the presenceof isopropanol for cofactor recycling, followed by incubation.
 6. Theprocess according to claim 5, wherein the concentration of isoalphaacids, i.e. the substrate, is maximized to increase the volumetricproductivity of the bioconversion.
 7. The process according to claim 5,wherein the concentration of the cofactor NADPH or NADP in the mixtureis minimized to improve the economics of the bioconversion.
 8. Theprocess according to claim 1, comprising adding the ketoreductase enzymeand NADPH or NADP to a mixture of isoalpha acids in the presence ofanother enzyme for cofactor recycling, followed by incubation.
 9. Theprocess according to claim 1, comprising adding a whole cellbiocatalyst, wherein the whole cell biocatalyst is an immobilizedmicroorganism expressing the gene which encodes a ketoreductase, to amixture of isoalpha acids followed by incubation.
 10. The processaccording to claim 1, comprising culturing a microorganism expressingthe gene which encodes the ketoreductase and adding isoalpha acids tothe culture.
 11. The process according to claim 1, comprising adding theketoreductase enzyme, wherein the ketoreductase is thermostable, to anextract of isoalpha acids wherein heat is applied, and the mixture isincubated.
 12. The process according to claim 1, wherein theketoreductase specifically reduces cis-isohumulone, cis-isocohumulone,and cis-isoadhumulone.
 13. The process according to claim 1, wherein theketoreductase specifically reduces trans-isohumulone,trans-isocohumulone, and trans-isoadhumulone.
 14. The process accordingto claim 1, comprising adding a mixture of 2 or more ketoreductaseenzymes in an amount effective to reduce a mixture of cis- andtrans-isoalpha acids, to their respective dihydroisoalpha acids.
 15. Theprocess according to claim 14, wherein the mixture of 2 or moreketoreductase enzymes produces a unique mixture of dihydroisoalpha acidsthat is distinct from that produced by chemical reducing agents, such assodium borohydride.
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. Aketoreductase enzyme which comprises the amino acid sequence of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:16 SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22.
 20. Theketoreductase enzyme according to claim 19, wherein the reductase enzymeor microorganism expressing a gene which encodes the reductase canoptionally have one or more differences at amino acid residues ascompared to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO;8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQID NO:20, or SEQ ID NO:22,
 21. The ketoreductase enzyme according toclaim 20, wherein the ketoreductase is 99, 95, 90, 85, 80, 75 or 70percent homologous to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:22.
 22. The process according to claim1, wherein the ketoreductase enzyme comprises the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22. 23.The process according to claim 22, wherein the ketoreductase enzyme ormicroorganism expressing a gene which encodes the ketoreductase canoptionally have one or more differences at amino acid residues ascompared to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQID NO:20, or SEQ ID NO:22.
 24. The process according to claim 23,wherein the ketoreductase is 99, 95, 90, 85, 80, 75 or 70 percenthomologous to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:22.